Research Article /Araştırma Makalesi Doi: https://doi.org/10.37696/nkmj.654542 e‐ISSN: 2587‐0262 THE EFFECTS OF L-NAME AND AGMATINE IN THE NUCLEUS ACCUMBENS CORE REGION ON MORPHINE WITHDRAWAL SYNDROME Nukleus Akumbens Core Bölgesinde L-NAME ve Agmatinin Morfin Yoksunluğuna Etkileri Mahluga JAFAROVA DEMİRKAPU1 , Hasan Raci YANANLI 2 , Elmar MAMMADOV2 , Ina DERVİSHİ2 , Ali KIRBAŞ2 , Şafak Recep YAŞAR2 , Tzemal SALİ2 , Mansur KURBANOĞLU2 , Merve ÇAĞLAR2 , Öykü USLU2 , Halil Eren SAKALLİ3 , Betilay TOPKARA4 All experimental procedures were approved by the local ethics committee MUHDEK (Approval No.21.2012.mar). 1 Tekirdag Namik Kemal University, Department of Pharmacology, Tekirdag, TURKEY. 2 Marmara University School of Medicine, Istanbul, TURKEY. 3Eastern Mediterranean University Faculty of Medicine, Famagusta, NORTH CYPRUS. 4Koç University School of Medicine, Istanbul, TURKEY. Abstract Aim: The mesocorticolimbic dopaminergic system, especially the nucleus accumbens, is an important region in opioid dependence and withdrawal. Studies have shown that nitric oxide synthase inhibitors modulate the development of tolerance to opioids, opioid dependence, and withdrawal. In this study, we aimed to investigate the effects of local injections of L-NAME and agmatine into the nucleus accumbens core (NAcc), one of the nucleus accumbens subregions on withdrawal signs and locomotor activity behavior during naloxone-induced withdrawal in morphine-dependent rats. Materials and Methods: Twenty-four adult Sprague-Dawley rats were used in the study. Morphine dependence was developed in all animals after guide cannula implantation into the NAcc region. On the last day of experiment, following bilateral L-NAME, agmatine or artificial cerebrospinal fluid (aCSF, control group) microinjections morphine withdrawal was induced by naloxone. Results: Local administration of agmatine and L-NAME into the NAcc significantly suppressed the jumping number during naloxone induced withdrawal. Local agmatine treatment significantly suppressed the score of teeth chattering, although the L-NAME did not change. No significant difference was observed in withdrawal symptoms such as wet dog shakes and defecation after local agmatine and L-NAME treatment. Agmatine increased stereotypic movements, but did not change locomotor activity behaviors such as ambulatory activity and total covered distance. Local administration of L-NAME into the NAcc did not increase stereotypic and ambulatory movements, and total covered distance during naloxone-induced withdrawal. Conclusion: These results suggest that inhibition of nitric oxide synthesis in NAcc plays a role in morphine withdrawal symptoms, but it is not responsible alone. Keywords: Nucleus accumbens; L-NAME; agmatine; morphine; withdrawal. Öz Amaç: Mezokortikolimbik dopaminerjik sistem, özellikle de nukleus akumbens bölgesi opioid bağımlılığı ve yoksunluğunda önemli bölgelerdendir. Yapılan çalışmalara göre nitrik oksit sentaz inhibitörleri opioidlere karşı tolerans gelişimini, opioid bağımlılığı ve yoksunluğunu değiştirmektedir. Biz bu çalışmada L-NAME ve agmatinin nukleus akumbens alt-bölgelerinden biri olan nukleus akumbens çekirdek (NAcc) bölgesine lokal enjeksiyonlarının morfin bağımlısı sıçanlarda naloksonla tetiklenen yoksunluk sırasında yoksunluk bulguları ve lokomotor aktivite davranışı üzerine etkilerini araştırmayı amaçladık. Materyal ve Metot: Çalışmada yirmi dört yetişkin Sprague-Dawley sıçanı kullanıldı. Tüm hayvanlarda morfin bağımlılığı NAcc bölgelerine kılavuz kanüller yerleştirildikten sonra geliştirildi. Deneyin son gününde bilateral L-NAME, agmatin veya aCSF (yapay beyin omurilik sıvısı; kontrol grubu) mikroenjeksiyonlarını takiben nalokson uygulanarak morfin yoksunluğu tetiklendi. Bulgular: NAcc bölgesine lokal uygulanan agmatin ve L-NAME morfin bağımlısı hayvanlarda nalokson sonrası sıçrama sayısını anlamlı olarak baskıladı. Lokal L-NAME tedavisi diş gıcırdatma skorunu değiştirmediği halde agmatin tedavisi anlamlı düzeyde baskıladı. Lokal L-NAME ve agmatin tedavisinden sonra ıslak köpek silkinmesi ve defekasyon gibi yoksunluk bulgularında anlamlı farklılık saptanmadı. NAcc bölgesine lokal enjekte edilen agmatin stereotipik hareketleri artırdığı halde ambulatuvar ve toplam kat edilen mesafe gibi lokomotor aktivite davranışlarında anlamlı değişiklik yapmadı. NAcc bölgesine lokal enjekte edilen L-NAME naloksonla tetiklenen yoksunluk sendromunda stereotipik hareketlerde, ambulatuvar hareketlerde ve toplam kat edilen mesafede artışa yol açmadı. Sonuç: Bu bulgular nitrik oksit üretiminin NAcc bölgesinde baskılanmasının morfin yoksunluk sendromunda rol oynadığını, fakat tek başına sorumlu olmadığını göstermektedir. Anahtar Kelimeler: Nukleus akumbens; L-NAME; agmatin; morfin; yoksunluk. Corresponding Author / Sorumlu Yazar: Article History / Makale Geçmişi: Hasan Raci YANANLI Date Received / Geliş Tarihi: 03.12.2019 Adres: Departmant of Pharmacology, Marmara University Date Accepted / Kabul Tarihi: 04.03.2020 School of Medicine, Hastane Yolu Street number 5, Istanbul, Maltepe, postal code: 34854, İstanbul / TURKEY. Namık Kemal Tıp Dergisi 2020; 8(1): 65 - 72 E-posta: [email protected] DEMİRKAPU et al. Namık Kemal Tıp Dergisi 2020; 8(1): 65 ‐ 72 INTRODUCTION Agmatine, a decarboxylated arginine, is produced from L-arginine by arginine Morphine is an opioid drug that leads to decarboxylase. It is synthesized in both the addiction after repeated exposure or chronic brain and spinal cord, stored in synaptic use, and to withdrawal when discontniued. The vesicles in heterogeneously distributed role of the mesocorticolimbic dopaminergic neurons, released from the axon end by Ca2+ system in opioid dependence and withdrawal is dependent depolarization, and inactivated by known1. The dopaminergic neuronal network, reuptake and degraded by agmatinase10,11. which has proven importance in reward- After release from the neuron, agmatine punishment-learning functions, extends from interacts with α2 adrenoceptors, NMDA, and the ventral tegmental area (VTA) to the the imidazoline receptors, which are known to be nucleus accumbens (NAc)2. The NAc, which important in dependence and withdrawal, as has an important role in learning and well as nicotinic receptors12. Agmatine also motivation, consists of two subregions, namely inhibits NOS13,14. For this reason, studies have the nucleus accumbens core (NAcc) and the been conducted considering that agmatine, like nucleus accumbens shell (NAcs). The NAcc NOS inhibitors, may be effective in opioid contributes to the basal ganglia motor functions, dependence and withdrawal15. In a study, and the NAcs contributes to viscero-endocrine agmatine inhibited morphine-induced functions. Additionally, glutamatergic NMDA physiologic dependence16. In another study, receptors are more distributed in the NAcc, and agmatine prevented withdrawal symptoms such glutamatergic AMPA and GABAergic receptors as jumping, wet dog shakes, tremor, abnormal are better distributed in the NAcs3. Nitric oxide posture, weight loss, defecation, ptosis, teeth (NO) is produced from L-arginine by nitric oxide chattering, and diarrhea in a dose-dependent synthase (NOS), and distributed in various manner17. tissues, including the brain. In addition to its peripheral effects, NO is thought to be a In our previous study, an NOS inhibitor and neuromodulator, or even a neurotransmitter4,5. agmatine suppressed withdrawal symptoms It has been proven previously that NOS after systemic administration18. The aim of our inhibitors and the NO/cGMP pathway modulate study was to investigate the effects of local the development of tolerance to opioids, opioid administration of L-NAME, a non-selective NOS dependence, and withdrawal6,7. NOS inhibitors inhibitor, and agmatine into the NAcc on suppressed signs such as teeth chattering, withdrawal signs and locomotor activity penile licking, diarrhoea, chewing, wet dog behavior during naloxone-induced morphine shakes, and grooming, and unaffected signs withdrawal syndrome. such as rearing, jumping, ptosis, rhinorrhoea, MATERIALS AND METHODS and irritability on touch in naloxone-precipitated 8 morphine withdrawal syndrome . In another Subjects study, NOS inhibitors caused an increase in Twenty-four (eight animals in each group) adult withdrawal symptoms such as exploratory male and female Sprague-Dawley rats (250- 9 locomotor activity . 350g) supplied from Marmara University Animal Center (DEHAMER) were used in the study. The rats were housed with a reversed 12 h 66 DEMİRKAPU et al. Namık Kemal Tıp Dergisi 2020; 8(1): 65 ‐ 72 light/dark cycle at 21 ± 3°C and 50 ± 5% animals were considered dependent on day humidity. There was unlimited access to 1220-22. On the 12th day of the experiment, standard rat chow and water. The experimental following bilateral parenchymal microinjections procedure is shown in Figure 1. All of L-NAME, agmatine or aCSF into the NAcc, experimental procedures were approved by the naloxone (2 mg/kg, i.p.) was administered in the local ethics committee MUHDEK (Approval 5th minute and morphine withdrawal was No.21.2012.mar). induced. After the naloxone injection, each rat was immediately placed into a locomotor cage Guide Cannula Implantation for 15 min. LMA measurement and morphine Stereotaxic surgery was conducted on all rats withdrawal signs such as jumping, teeth under 100 mg/kg ketamine and 10 mg/kg chattering, wet dog shakes, and defecation xylazine (i.p.) anesthesia. According to the were simultaneously evaluated for the same standard stereotaxic surgery